Process for the specific isotopic labeling of methyl groups of val, leu and ile

ABSTRACT

The invention relates to a process for the specific isotopic labeling of Valine, Leucine and Isoleucine amino acids. The process of the invention uses a 2-alkyl-2-hydroxy-3-oxobutanoic acid in which the alkyl substituent in position 2 is ethyl or methyl. The invention can be used for the analysis of proteins, in particular by NMR.

The invention relates to a process for the specific isotopic labeling ofValine, Leucine and Isoleucine amino acids and more particularly for thestereospecific labeling of methyl groups of Leucine and Valine as wellas specific labeling of the γ2 methyl groups of Isoleucine in proteinsand biomolecular assemblies.

It also relates to specifically methyl labeled2-hydroxy-2-methyl-3-oxobutanoic acid and2-ethyl-2-hydroxy-3-oxobutanoic acid (named as acetolactate derivativesin the following) used in this process and to a process formanufacturing such specifically methyl labeled acetolactate derivatives.

NMR spectroscopy is an established and powerful method for structuraldetermination and to map biomolecular interactions in complexes withaffinities ranging from low nanomolar range to a few millimolar range.

But poor resolution in NMR spectra remains a major limiting factor tothe application of this method to large molecular assemblies such asproteins of molecular weight up to 1 megadalton, even though recentprogress in NMR spectroscopy of high molecular weight proteins have beenmade. This progress is strongly connected to the development of newisotopic labeling schemes. The combination of selected protonation ofmethyl groups in fully perdeuterated proteins with transverse relaxationoptimized methyl spectroscopy (methyl-TROSY) has allowed local structureand dynamic proteins assemblies of up to 1 megadalton to be studied bysolution NMR spectroscopy.

Such labeling protocols rely on the addition of specific [¹H¹³C] methyllabeled biosynthetic precursors as the sole proton source in aperdeuterated culture medium.

This approach provides a high level of methyl protonation withoutdetectable isotopic scrambling.

Valine (Val) and Leucine (Leu) and Isoleucine (Ile) are amino acids ofgreat interest as their methyl groups account for more than 50% of thetotal methyl probes available in proteins.

Protonation of Leu and Val methyl groups in perdeuterated proteins iscommonly achieved using methyl protonated 2-oxo-3-methylbutanoic acid(named α-ketoisovalerate in the following) an intermediate in thebiosynthesis of such amino acids.

However, in particular for large proteins of 30 kDa to 1 megaDa, thislabeling strategy can result in overcrowded [¹H¹³C]-correlation spectradue to the sheer number of NMR-visible methyl-probes. Overlap in NMRspectra can greatly complicate the measurement of site-specificstructural or relaxion parameters.

The invention aims to overcome the drawbacks of this method by providinga process for specific isotopic labeling of Valine, Leucine andIsoleucine amino acids and more particularly for the labeling of methylgroups of Leucine, Valine and Isoleucine in recombinant perdeuteratedproteins.

This process offers a significant enhancement in the resolution andsensitivity of [¹³C¹H]-methyl TROSY spectra and extends the capacity fordetecting long-range structurally meaningful distance restraints inlarge proteins. This method is particularly useful for investigation ofmolecular interactions within large biomolecular assemblies.

Furthermore this process allows for proteins of little and medium size(i.e. of less than 100 kDa) a significant improvement for thestereospecific assignment of methyl groups of the Leucine and Valineaminoacids compared to precedent methods using mixture of unlabeled andlabeled precursors (glucose or pyruvate). This last method is describedin D. Neri, T. Szyperski, G. Otting, H. Senn, K. Wüthrich, Biochemistry1989, 28, 7510-7516.

For this end, the invention proposes a process for the specific isotopiclabeling of amino acids selected from the group consisting of Val, Leu,and Ile, and more particularly for the stereospecific labeling of methylgroups of Leu and Val as well as specific labeling of the γ2 methylgroups of Isoleucine in proteins and biomolecular assemblies comprisingthe following step a):

introducing, in a medium containing bacteria overexpressing a protein, a2-alkyl-2-hydroxy-3-oxobutanoic acid, wherein the alkyl substituent inposition 2 is ethyl or methyl, also called acetolactate derivatives inthe following and having the following formula:

wherein:

-   -   X is ¹H or ²H (D),    -   each Y is independently from the others ¹²C or ¹³C,    -   R¹ is a methyl group in which the carbon atom is ¹³C or ¹²C and        the hydrogen atoms are independently from each other ¹H or ²H        (D),    -   R² is either a methyl group in which the carbon atom is ¹³C or        ¹²C and the hydrogen atoms are independently from each other ¹H        or ²H (D), or an ethyl group in which the carbon atoms are        independently from each other ¹³C or ¹²C and the hydrogen atoms        are independently from each other ¹H or ²H (D),        at the provisos that:

1) the hydrogen atoms of the acetolactate derivative of Formula I arenot all, at the same time, either ¹H or ²H (D),

2) the hydrogen atoms of R¹ and the hydrogen atoms of R² are not all, atthe same time, either ¹H or ²H (D).

In Formula I, X is an exchangeable hydrogen which can be ¹H or ²Hdepending on the nature of the solvent.

This process furthermore comprises the following steps:

b) overexpression of the protein by the bacteria contained in themedium, and

c) purification of the protein.

In a first preferred embodiment of the process of labeling of theinvention, the acetolactate derivative has the Formula I in which:

-   -   R¹ is chosen among the following groups:    -   ¹²CH₃, ¹²CD₃, ¹³CH₃, ¹³CD₃, ¹³CHD₂, ¹³CH2D,    -   R² is chosen among the following groups:    -   ¹²CH₃, ¹²CD₃, ¹³CH₃, ¹³CD₃, ¹³CHD₂, ¹³CH₂D, ¹²CH₃ ¹²CD₂, ¹²CD₃        ¹²CD₂, ¹³CH₃ ¹²CD₂, ¹³CH₃ ¹³CD₂, ¹³CD₃ ¹³CD₂, ¹³CHD₂ ¹³CD₂,        ¹³CH₂D¹³CD₂, ¹³CHD₂ ¹²CD₂, ¹³CH₂D¹²CD₂,    -   each Y is independently from the others ¹²C or ¹³C,        at the provisos that:

1) the hydrogen atoms of the acetolactate derivative of Formula I arenot all, at the same time, either ¹H or ²H (D),

2) the hydrogen atoms of R¹ and the hydrogen atoms of R² are not all, atthe same time, either ¹H or ²H (D).

In a second preferred embodiment of the process of labeling of theinvention, the acetolactate derivative is selected in the group ofcompounds having the following formulae:

-   4=2-hydroxy-2-(¹³C)methyl-3-oxo-4(²H₃)butanoic acid,-   5=2-hydroxy-2-(²H₃)methyl-3-oxo-4(¹³C)butanoic acid,-   6=2-(²H₅)ethyl-2-hydroxy-3-oxo-4-(¹³C)methylbutanoic acid,-   9=1,2,3,4-(¹³C)-2-(²H₅)ethyl-2-hydroxy-3-oxobutanoic acid,-   21=1,2,3-(¹³C)-2-(¹³C)methyl-2-hydroxy-3-oxo-4-(²H₃)butanoic Acid,-   22=1,2,3,4-(¹³C)-2-(²H₃)methyl-2-hydroxy-3-oxobutanoic acid,-   24=1,2,3-(¹³C)-2-(1′-(²H₂),    ¹³C₂)ethyl)-2-hydroxy-3-oxo-4-(²H₃)butanoic acid,-   36=3,4-(¹³C)-2-(¹³C)methyl-2-hydroxy-3-oxo-4-(²H₃)butanoic acid,-   37=3,4-(¹³C)-2-(²H₃,¹³C)methyl-2-hydroxy-3-oxobutanoic acid-   40=3,4-(¹³C)-2-(¹³C)methyl-2-hydroxy-3-oxobutanoic acid,-   45=3,4-(¹³C)-2-(1′-(²H₂),¹³C₂)ethyl-2-hydroxy-3-oxo-4-(²H₃)butanoic    acid,-   46=3,4-(¹³C)-2-(²H₅,¹³C₂)ethyl-2-hydroxy-3-oxobutanoic acid,-   49=3,4-(¹³C)-2-(1′-(²H₂),¹³C₂)ethyl-2-hydroxy-3-oxobutanoic acid.

In a third preferred embodiment of the process of labeling of theinvention, the acetolactate derivative is selected in the group ofcompounds having the following formulae:

-   13=2-hydroxy-2-(²H₂,¹³C)methyl-3-oxo-4-(²H₃)butanoic acid,-   14=2-hydroxy-2-(²H₃)methyl-3-oxo-4-(²H₂,¹³C)butanoic acid,-   15=2-(²H₅)ethyl-2-hydroxy-3-oxo-4-(²H₂,¹³C)butanoic acid,-   34=2-(1′-(²H₂),2′-(²H),2′-(¹³C))ethyl-2-hydroxy-3-oxo-4-(²H₃)butanoic    acid.

In a fourth preferred embodiment of the process of labeling of theinvention, acetolactate derivative is selected in the group of compoundsof the following formulae:

-   10=2-hydroxy-2-(²H,¹³C)methyl-3-oxo-4-(²H₃)butanoic acid,-   11=2-hydroxy-2-(²H₃)methyl-3-oxo-4-(²H,¹³C)butanoic acid,-   12=2-(²H₅)ethyl-2-hydroxy-3-oxo-4-(²H,¹³C)butanoic acid.

The invention also proposes a compound of the following formula I-1:

wherein:

-   -   X is ¹H or ²H (D),    -   each Y is independently from the others ¹²C or ¹³C,    -   R¹ is a methyl group in which the carbon atom is ¹³C or ¹²C and        the hydrogen atoms are independently from each other ¹H or ²H        (D),    -   R² is either a methyl group in which the carbon atom is ¹³C or        ¹²C and the hydrogen atoms are independently from each other ¹H        or ²H (D), or an ethyl group in which the carbon atoms are        independently from each other ¹³C or ¹²C and the hydrogen atoms        are independently from each other ¹H or ²H (D),        at the provisos that:

1) the hydrogen atoms of the acetolactate derivative of Formula I arenot all, at the same time, either ¹H or ²H (D),

2) the hydrogen atoms of R¹ and the hydrogen atoms of R² are not all, atthe same time, either ¹H or ²H (D),

3) when all the hydrogen atoms of R¹ are ²H, then the carbon atoms, informula I-1, are not all, at the same time, ¹²C.

The preferred compounds of the invention are selected from the groupconsisting of the compounds having the formula I-1 in which:

-   -   R¹ is chosen among the following groups:    -   ¹²CH₃, ¹²CD₃, ¹³CH₃, ¹³CD₃, ¹³CHD₂, ¹³CH₂D,    -   R² is chosen among the following groups:    -   ¹² _(CH) ₃, ¹²CD₃, ¹³CH₃, ¹³CD₃, ¹³CHD₂, ¹³CH₂D, ¹²CH₃ ¹²CD₂,        ¹²CD₃ ¹²CD₂, ¹³CH₃ ¹²CD₂, ¹³CH₃ ¹³CD₂, ¹³CD₃ ¹³CD₂, ¹³CHD₂        ¹³CD₂, ¹³CH₂D¹³CD₂, ¹³CHD₂ ¹²CD₂, ¹³CH₂D¹²CD₂,    -   each Y is independently from the others ¹²C or ¹³C,        at the provisos that:

1) the hydrogen atoms of the acetolactate derivative of Formula I arenot all, at the same time, either ¹H or ²H (D),

2) the hydrogen atoms of R¹ and the hydrogen atoms of R² are not all, atthe same time, either ¹H or ²H (D), and

3) when all the hydrogen atoms of R¹ are ²H, then the carbon atoms, informula I-1, are not all, at the same time, ¹²C.

It is to be noted that the active part of the acetolactate derivativesof formula I is, in fact the anion of the following formula:

wherein:

X, Y, R¹ and R² are as defined for the compounds of formulae I and I-1.

In a first preferred embodiment, the compound of formula I-1 of theinvention is selected among the compounds of the following formulae:

-   4=2-hydroxy-2-(¹³C)methyl-3-oxo-4(²H₃)butanoic acid,-   5=2-hydroxy-2-(²H₃)methyl-3-oxo-4(¹³C)butanoic acid,-   6=2-(²H₅)ethyl-2-hydroxy-3-oxo-4-(¹³C)methylbutanoic acid,-   9=1,2,3,4-(¹³C)-2-(²H₅)ethyl-2-hydroxy-3-oxobutanoic acid,-   21=1,2,3-(¹³C)-2-(¹³C)methyl-2-hydroxy-3-oxo-4-(²H₃)butanoic Acid,-   22=1,2,3,4-(¹³C)-2-(²H₃)methyl-2-hydroxy-3-oxobutanoic acid,-   24=1,2,3,4-(¹³C)-2-(1′-(²H₂),    ¹³C₂)ethyl)-2-hydroxy-3-oxo-4-(²H₃)butanoic acid,-   36=3,4-(¹³C)-2-(¹³C)methyl-2-hydroxy-3-oxo-4-(²H₃)butanoic acid,-   37=3,4-(¹³C)-2-(²H₃,¹³C)methyl-2-hydroxy-3-oxobutanoic acid-   40=3,4-(¹³C)-2-(¹³C)methyl-2-hydroxy-3-oxobutanoic acid,-   45=3,4-(¹³C)-2-(1′-(²H₂),¹³C₂)ethyl-2-hydroxy-3-oxo-4-(²H₃)butanoic    acid,-   46=3,4-(¹³C)-2-(²H₅,¹³C₂)ethyl-2-hydroxy-3-oxobutanoic acid,-   49=3,4,-(¹³C)-2-(1′-(²H₂),¹³C₂)ethyl-2-hydroxy-3-oxobutanoic acid.

In a second preferred embodiment, the compound of formula I-1 of theinvention is selected among the compounds of the following formulae:

-   13=2-hydroxy-2-(²H₂,¹³C)methyl-3-oxo-4-(²H₃)butanoic acid,-   14=2-hydroxy-2-(²H₃)methyl-3-oxo-4-(²H₂,¹³C)butanoic acid,-   15=2-(²H₅)ethyl-2-hydroxy-3-oxo-4-(²H₂,¹³C)butanoic acid,-   34=2-(1′-(²H₂),2′-(²H),2′-(¹³C))ethyl-2-hydroxy-3-oxo-4-(²H₃)butanoic    acid.

In a third preferred embodiment, the compound of formula I-1 of theinvention is selected among the compounds of the following formulae:

-   10=2-hydroxy-2-(²H,¹³C)methyl-3-oxo-4-(²H₃)butanoic acid,-   11=2-hydroxy-2-(²H₃)methyl-3-oxo-4-(²H,¹³C)butanoic acid,-   12=2-(²H₅)ethyl-2-hydroxy-3-oxo-4-(²H,¹³C)butanoic acid.

The invention also proposes a process for manufacturing a compound ofthe following formula I:

comprising the following steps:

a) alkylation with a methyl group in which the carbon atom is ¹³C or ¹²Cand the hydrogen atoms are independently from each other ¹H or ²H (D),or an ethyl group in which the carbon atoms are independently from eachother ¹³C or ¹²C and the hydrogen atoms are independently from eachother ¹H or ²H (D), of a 3-oxobutanoate derivative having its hydroxylgroup in position 1 protected by a protecting group, preferably a methylor ethyl group,

b) hydroxylation of the compound obtained in step a),

c) optionally deprotection and exchange of the desired ¹H atoms by ²Hatoms,

wherein step b) of hydroxylation is carried out by usingdimethyldioxirane in presence of Nickel (II) ions.

Furthermore, the invention also proposes processes for analysingproteins by NMR.

In a first embodiment, this process comprises a step of labeling theproteins to be analysed by the labeling process of the invention.

In a second embodiment, this process comprises a step of labeling theproteins to be analysed with a compound of the following formula I:

wherein:

-   -   X is ¹H or ²H (D),    -   each Y is independently from the others ¹²C or ¹³C,    -   R¹ is a methyl group in which the carbon atom is ¹³C or ¹²C and        the hydrogen atoms are independently from each other ¹H or ²H        (D),    -   R² is either a methyl group in which the carbon atom is ¹³C or        ¹²C and the hydrogen atoms are independently from each other ¹H        or ²H (D), or an ethyl group in which the carbon atoms are        independently from each other ¹³C or ¹²C and the hydrogen atoms        are independently from each other ¹H or ²H (D),        at the provisos that:

1) the hydrogen atoms of the acetolactate derivative of Formula I arenot all, at the same time, either ¹H or ²H (D),

2) the hydrogen atoms of R¹ and the hydrogen atoms of R² are not all, atthe same time, either ¹H or ²H (D).

In a third embodiment, this process comprises a step of labeling theproteins to be analysed with a compound of the following formula I-1:

wherein:

-   -   X is ¹H or ²H (D),    -   each Y is independently from the others ¹²C or ¹³C,    -   R¹ is a methyl group in which the carbon atom is ¹³C or ¹²C and        the hydrogen atoms are independently from each other ¹H or ²H        (D),    -   R² is either a methyl group in which the carbon atom is ¹³C or        ¹²C and the hydrogen atoms are independently from each other ¹H        or ²H (D), or an ethyl group in which the carbon atoms are        independently from each other ¹³C or ¹²C and the hydrogen atoms        are independently from each other ¹H or ²H (D),        at the provisos that:

1) the hydrogen atoms of the acetolactate derivative of Formula I arenot all, at the same time, either ¹H or ²H (D),

2) the hydrogen atoms of R¹ and the hydrogen atoms of R² are not all, atthe same time, either ¹H or ²H (D),

3) when all the hydrogen atoms of R¹ are ²H, then the carbon atoms, informula I-1, are not all, at the same time, ¹²C.

The invention will be better understood and other features andadvantages thereof will be more apparent when reading the followingdescription.

In the description of the invention, the following terms have thefollowing meanings:

-   -   Val designates amino acids Valine,    -   Leu designates amino acids Leucine,    -   Ile designates amino acids Isoleucine,    -   C designates ¹³C,    -   C designates ¹²C,    -   D designates ²H,    -   H designates ¹H,    -   proR, proS: the methyl groups on the γ and β carbons of        respectively unlabeled Leu and Val aminoacids, are not different        and consequently the γ and β carbon of respectively unlabeled        Leu and Val aminoacids are not chiral. But when the groups R¹        and R² are not labeled in the same manner in the acetolactate        derivatives of formula I, the resulting methyl groups on the γ        and β carbon of respectively Leu and Val are differently labeled        and due to this difference the γ and β carbon of respectively        Leu and Val become chiral. These methyl groups are designated as        proR when labeling gives rise to a R configuration and as proS        when labeling gives rise to a S configuration.    -   “Acetolactate derivative” designates compounds of formula I and        their corresponding esters, preferably methyl esters or ethyl        esters.    -   Biomolecular assemblies: molecules containing proteins and other        groups.

The process of labeling of the invention is based on the use ofacetolactate derivatives which have methyl or ethyl groups specificallylabeled. These acetolactate derivatives are introduced in a mediumcontaining bacteria overexpressing a protein or proteins of interest.

Otherwise stated, the process of the invention is based on thestereospecific rearrangement of labeled or unlabeled alkyl groups inacetolactate derivatives occurring in vivo in the early steps of Leu,Val and Ile biogenesis.

The proposed process of the invention for the specific isotopic labelingof methyl groups of amino acids, which are selected from the groupconsisting of Valine (Val), Leucine (Leu), and Isoleucine (Ile), inproteins comprises the following step a):

introducing, in a medium containing bacteria overexpressing a protein, aderivative of acetolactate having the following formula:

wherein:

-   -   X is an exchangeable hydrogen being ¹H or ²H (D) depending on        the nature of the solvent, i.e. X is ¹H when the solvent is H₂O        and X is ²H when the solvent is D₂O,    -   each Y is independently from the others ¹²C or ¹³C,    -   R¹ is a methyl group in which the carbon atom is ¹³C or ¹²C and        the hydrogen atoms are independently from each other ¹H or ²H        (D),    -   R² is either a methyl group in which the carbon atom is ¹³C or        ¹²C and the hydrogen atoms are independently from each other ¹H        or ²H (D), or an ethyl group in which the carbon atoms are        independently from each other ¹³C or ¹²C and the hydrogen atoms        are independently from each other ¹H or ²H (D),        at the provisos that:

1) the hydrogen atoms of the acetolactate derivative of Formula I arenot all, at the same time, either ¹H or ²H (D),

2) the hydrogen atoms of R¹ and the hydrogen atoms of R² are not all, atthe same time, either ¹H or ²H (D).

Then, this process comprises a step of culture of the medium foroverexpressing the protein(s) of interest and the purification andisolation of the protein(s).

Preferably, the acetolactate derivatives of formula I are selected inthe group consisting of the compounds of the following formulae:

-   -   R¹ is chosen among the following groups:    -   ¹²CH₃, ¹²CD₃, ¹³CH₃, ¹³CD₃, ¹³CHD₂, ¹³CH₂D,    -   R² is chosen among the following groups:    -   ¹²CH₃, ¹²CD₃, ¹³CH₃, ¹³CD₂, ¹³CHD₂, ¹³CH₂D, ¹²CH₃ ¹²CH₂, ¹²CD₃        ¹²CD₂, ¹³CH₃ ¹²CD₂, ¹³CH₃ ¹³CD₂, ¹³CD₃ ¹³CD₂, ¹³CHD₂ ¹³CD₂,        ¹³CH₂D¹³CD₂, ¹³CHD₂ ¹²CD₂, ¹³CH₂D¹²CD₂,    -   each Y is independently from the others ¹²C or ¹³C,        at the provisos that:

1) the hydrogen atoms of the acetolactate derivative of Formula I arenot all, at the same time, either ¹H or ²H (D),

2) the hydrogen atoms of R¹ and the hydrogen atoms of R² are not all, atthe same time, either ¹H or ²H (D).

These acetolactate derivatives have the following formulae 1-57 inwhich:

-   -   (¹³C) methyl means that the carbon atoms of the methyl groups is        ¹³C,    -   (¹³C₂) ethyl means that the two carbon atoms of the ethyl groups        are ¹³C,    -   ²H_(n), in which n=1, 2, 3, 4 or 5 means that the n hydrogen        atoms are ²H, and    -   U means that all the carbon atoms are ¹³C:

-   1=2-hydroxy-2-methyl-3-oxo-4-(²H₃)butanoic acid,

-   2=2-hydroxy-2-(²H₃)methyl-3-oxobutanoic acid,

-   3=2-(²H₅)ethyl-2-hydroxy-3-oxobutanoic acid,

-   4=2-hydroxy-2-(¹³C)methyl-3-oxo-4(²H₃)butanoic acid,

-   5=2-hydroxy-2-(²H₃)methyl-3-oxo-4(¹³C)butanoic acid,

-   6=2-(²H₅)ethyl-2-hydroxy-3-oxo-4-(¹³C)methylbutanoic acid,

-   7=U-(¹³C)-2-hydroxy-2-methyl-3-oxo-4(²H₃)butanoic acid,

-   8=U-(¹³C)-2-hydroxy-2-(²H₃)methyl-3-oxobutanoic acid,

-   9=1,2,3,4-(¹³C)-2-(²H₅)ethyl-2-hydroxy-3-oxobutanoic acid,

-   10=2-hydroxy-2-(²H,¹³C)methyl-3-oxo-4-(²H₃)butanoic acid,

-   11=2-hydroxy-2-(²H₃)methyl-3-oxo-4-(²H,¹³C)butanoic acid,

-   12=2-(²H₅)ethyl-2-hydroxy-3-oxo-4-(²H,¹³C)butanoic acid,

-   13=2-hydroxy-2-(²H₂,¹³C)methyl-3-oxo-4-(²H₃)butanoic acid,

-   14=2-hydroxy-2-(²H₃)methyl-3-oxo-4-(²H₂,¹³C)butanoic acid,

-   15=2-(²H₅)ethyl-2-hydroxy-3-oxo-4-(²H₂,¹³C)butanoic acid,

-   16=U-(¹³C)-2-hydroxy-2-(²H₂)methyl-3-oxo-4-(²H₃)butanoic acid,

-   17=U-(¹³C)-2-hydroxy-2-(²H₃)methyl-3-oxo-4-(²H₂)butanoic acid,

-   18=1,2,3,4-(¹³C)-2-(²H₅)ethyl-2-hydroxy-3-oxo-4-(²H₂)butanoic acid,

-   19=2-(1′-(²H₂)ethyl-2-hydroxy-3-oxo-4-(²H₃)butanoic acid,

-   20=2-((1′-(²H₂),2′-(¹³C))ethyl-2-hydroxy-3-oxo-4-(²H₃)butanoic acid,

-   21=1,2,3-(¹³C)-2-(¹³C)methyl-2-hydroxy-3-oxo-4-(²H₃)butanoic acid,

-   22=1,2,3,4-(¹³C)-2-(²H₃)methyl-2-hydroxy-3-oxobutanoic acid,

-   23=U-(¹³C)-2-(²H₅)ethyl-2-hydroxy-3-oxobutanoic acid,

-   24=1,2,3-(¹³C)-2-(1′-(²H₂),¹³C₂)ethyl)-2-hydroxy-3-oxo-4-(²H₃)butanoic    acid,

-   25=U-(¹³C)-2-(1′-(²H₂))ethyl-2-hydroxy-3-oxo-4-(²H₃)butanoic acid,

-   26=U-(¹³C)-2-(²H)methyl-2-hydroxy-3-oxo-4-(²H₃)butanoic acid,

-   27=U-(¹³C)-2-(²H₃)methyl-2-hydroxy-3-oxo-4-(²H)butanoic acid,

-   28=U-(¹³C)-2-((²H₅)ethyl)-2-hydroxy-3-oxo-4-(²H)butanoic acid,

-   29=1,2,3-(¹³C)-2-(²H,¹³C)methyl-2-hydroxy-3-oxo-4-(²H₃)butanoic    acid,

-   30=1,2,3,4-(¹³C)-2-(²H₃)methyl-2-hydroxy-3-oxo-4-(²H)butanoic acid,

-   31=1,2,3,4-(¹³C)-2-(²H₅)ethyl-2-hydroxy-3-oxo-4-(²H)butanoic acid,

-   32=U-(¹³C)-2-(1′-(²H₂),2′-(²H))ethyl-2-hydroxy-3-oxo-4-(²H₃)butanoic    acid,

-   33=1,2,3-(¹³C)-2-(2′-(²H),¹³C₂)ethyl-2-hydroxy-3-oxo-4-(²H₃)butanoic    acid,

-   34=2-(1′-(²H₂),2′-(²H),2′-(¹³C))ethyl-2-hydroxy-3-oxo-4-(²H₃)butanoic    acid,

-   35=1,2,3-(¹³C)-2-(²H₂,¹³C)methyl-2-hydroxy-3-oxo-4-(²H₃)butanoic    acid,

-   36=3,4-(¹³C)-2-(¹³C)methyl-2-hydroxy-3-oxo-4-(²H₃)butanoic acid,

-   37=3,4-(¹³C)-2-(²H₃,¹³C)methyl-2-hydroxy-3-oxobutanoic acid,

-   38=3,4-(¹³C)-2-(²H₂,¹³C)methyl-2-hydroxy-3-oxo-4-(²H₃)butanoic acid,

-   39=3,4-(¹³C)-2-(²H,¹³C)methyl-2-hydroxy-3-oxo-4-(²H₃)butanoic acid,

-   40=3,4-(¹³C)-2-(¹³C)methyl-2-hydroxy-3-oxobutanoic acid,

-   41=3,4-(¹³C)-2-(²H,¹³C)methyl-2-hydroxy-3-oxo-4-(²H)butanoic acid,

-   42=3,4-(¹³C)-2-(²H₂,¹³C)methyl-2-hydroxy-3-oxo-4-(²H₂)butanoic acid,

-   43=3,4-(¹³C)-2-(²H₃,¹³C)methyl-2-hydroxy-3-oxo-4-(²H₂)butanoic acid,

-   44=3,4-(¹³C)-2-(²H₃,¹³C)methyl-2-hydroxy-3-oxo-4-(²H)butanoic acid,

-   45=3,4-(¹³C)-2-(1′-(²H₂),¹³C₂)ethyl-2-hydroxy-3-oxo-4-(²H₃)butanoic    acid,

-   46=3,4-(¹³C)-2-(²H₅,¹³C₂)ethyl-2-hydroxy-3-oxobutanoic acid,

-   47=3,4-(¹³C)-2-(1′-(²H₂),2′-(²H),¹³C₂)ethyl-2-hydroxy-3-oxo-4-(²H₃)butanoic    acid,

-   48=3,4-(¹³C)-2-(1′-(²H₂),2′-(²H₂),¹³C₂)ethyl-2-hydroxy-3-oxo-4-(²H₃)butanoic    acid,

-   49=3,4-(¹³C)-2-(1′-(²H₂),¹³C₂)ethyl-2-hydroxy-3-oxobutanoic acid,

-   50=3,4-(¹³C)-2-(1′-(²H₂),2′-(²H₂),¹³C₂)ethyl-2-hydroxy-3-oxo-4-(²H₂)butanoic    acid,

-   51=3,4-(¹³C)-2-(1′-(²H₂)-2′-(²H),¹³C₂)ethyl-2-hydroxy-3-oxo-4-(²H)butanoic    acid,

-   52=3,4-(¹³C)-2-(2H₅,¹³C₂)ethyl-2-hydroxy-3-oxo-4-(²H₂)butanoic acid,

-   53=3,4-(¹³C)-2-(2H₅,¹³C₂)ethyl-2-hydroxy-3-oxo-4-(²H)butanoic acid,

-   54=U-(¹³C)-2-(²H₅)ethyl-2-hydroxy-3-oxo-4-(²H₂)butanoic acid,

-   55=U-(¹³C)-2-(²H₅)ethyl-2-hydroxy-3-oxo-4-(²H)butanoic acid,

-   56=1,2,3-(¹³C)-2-(1′-(²H₂),2′-(²H₂),¹³C₂)ethyl-2-hydroxy-3-oxo-4-(²H₃)butanoic    acid,

-   57=U-(¹³C)-2-(1′-(²H₂),2′-(²H₂))ethyl-2-hydroxy-3-oxo-4-(²H₃)butanoic    acid.

In order to better illustrate the formulae of the compounds of theinvention, the developed formulae of compounds 1-18 are given below.

In these formulae, X designates ²H or ¹H, D designates ²H, C designates¹³C and C designates ¹²C:

More specifically, when the compound of formula 1 is used in the processof the invention, the protonated methyl groups are proS methyl groups ofValine (γ2 methyl) and proS methyl groups of Leucine (δ2 methyl) and are¹²CH₃.

Thus this compound enables to perform structural studies of proteinscontaining Leu and Val amino acids. The labeling with this compoundenables to determine the stereospecificity of the amino acids Val andLeu and also enables to detect weak as low as 0.05 Hz, dipolar andscalar interactions in proteins of less than 30 kDa.

This compound of formula 1 (2-hydroxy-2-methyl-3-oxo-4-(²H₃)-butanoicacid) enables to label amino acids Leu and Val incorporated in proteinsin the following manner:

When this compound of formula 1 is used to produce human ubiquitine andprotein G of streptoccocus (GB3) using E. coli cells in a deuterated M9medium using 300 mg/l of this compound of formula 1, only the proSmethyl groups of Val and Leu are protonated in these proteins and arethen observable in the NMR 1D spectra of the obtained compounds.

The compound of formula 2 (2-hydroxy-2-(²H₃)methyl-3-oxobutanoic acid)enables to stereospecifically label with ¹²CH₃ proR methyl groups of Leuand Val. This compound enables to perform structural studies,stereospecific attribution and detection of weak dipolar and scalarinteractions by NMR in proteins of less than 30 kDa.

The stereospecific labeling of these methyl groups with compound 2 givesthe following labeling of amino acids Leu and Val:

In NMR 1D spectra of proteins obtained by culture of a medium containingthis compound of formula 2, only proR methyl groups of Leucine andValine, more precisely the γ1 methyl group of Valine and the δ1 methylgroup of Leucine are observable.

The compound of formula 3 enables to specifically label with ¹²CH₃ γ2methyl groups of Ile. This compound enables to perform structuralstudies, specific attribution and detection of weak dipolar and scalarinteractions by NMR in proteins of less than 30 kDa.

The labeling of the γ2 methyl groups of Ile with the compound of formula3 (2-(²H₅)ethyl-2-hydroxy-3-oxobutanoic acid) is shown in the followingschema:

It enables to perform structural studies, by liquid NMR, of proteins andbiomolecular assemblies.

The compound of formula 4 enables to stereospecifically label with ¹³CH₃proS methyl groups of Valine (γ2 methyl) and proS methyl groups ofLeucine (δ2 methyl). It enables to perform structural studies by liquidNMR of proteins and large biomolecular assemblies.

The stereospecific labeling of proS methyl groups of Valine and Valinewith the compound of formula 4(2-hydroxy-2-(¹³C)methyl-3-oxo-4(²H₃)butanoic acid) is shown to thefollowing schema:

When the compound of formula 4 is used for stereospecifically labelingTET2 (U-[²H], U-[¹²C], Leu/Val-[¹³C¹H3]^(proS) TET2) a dodecamericprotease of 468 kDa and malate synthase G (MSG) (U-[²H], U-[¹²C],Leu/Val-[¹³C¹H3]^(proS) MSG) produced using E. coli cells in adeuterated M9 medium in presence of 300 mg/L of the compound of formula4, the methyl-TROSY spectra of the obtained compounds show that onlyhalf of the resonances of the methyl groups are observable.

The compound of formula 5 (2-hydroxy-2-(²H₃)methyl-3-oxo-4(¹³C)butanoicacid) enables to stereospecifically label with ¹³CH₃ proR methyl groupof the Valines (γ1 methyl) and proR methyl group of the Leucines (δ1methyl).

It enables to perform structural studies by liquid NMR of proteins andlarge biomolecular assemblies.

The labeling with this compound of formula 5 leads to labeled aminoacids Leucine and Valine as shown to the following schema:

When this compound of formula 5 is used for obtaining TET2 (U-[²H],U-[¹²C], Leu/Val-[¹³C¹H₃]^(proR) TET2), a dodecameric protease of 468kDa produced using E. coli cells in a deuterated M9 medium in presenceof 300 mg/L of the compound of formula 5, the methyl-TROSY spectra ofthe obtained compound shows only half of the resonance of the methylgroups as compared to the spectra of TET2 produced from isovaleratewhich is protonated on the two methyl groups.

The compound of formula 6(2-(²H₅)ethyl-2-hydroxy-3-oxo-4(¹³C)methylbutanoic acid) enables tolabel with ¹³CH_(3 γ2) methyl groups of Isoleucine.

It enables to perform structural studies by liquid NMR of proteins andlarge biomolecular assemblies.

This compound leads to (L)-Isoleucine labeled as shown in the followingschema.

The methyl-TROSY spectra of TET2 (U-[²H], U-[¹²C], Ile-[¹³C¹H₃]^(γ2)TET2), a dodecameric protease of 468 kDa produced in a deuterated M9medium in presence of 300 mg/L of the compound of formula 6 shows onlyγ2 methyl groups of Isoleucine.

The compound of formula 7(U-(¹³C)-2-hydroxy-2-methyl-3-oxo-4(²H₃)butanoic acid) enables tostereoselectively assign the proS methyl groups of Valine (γ2 methyl)and proS methyl groups of Leucine (δ2 methyl) in proteins and largebiomolecular assemblies by liquid NMR.

This compound leads to the following labeling of Leucine and Valine:

The compound of formula 8 (U-(¹³C)-2-hydroxy-2-(²H₃)methyl-3-oxobutanoicacid) enables stereo selectively assign the proR methyl groups of Valine(γ1 methyl) and proR methyl groups of Leucine (δ1 methyl) in proteinsand large biomolecular assemblies by liquid NMR.

The compound of formula 8 gives the following labeling of Leucine andValine amino acids in proteins and biomolecular assemblies:

The compound of formula 9(1,2,3,4-(¹³C)-2-(²H₅)ethyl-2-hydroxy-3-oxobutanoic acid) enables toassign the γ2 methyl groups of Isoleucine in proteins and biomolecularassemblies by liquid NMR.

Isoleucine is labeled as shown below:

The compound of formula 10(2-hydroxy-2-(²H,¹³C)methyl-3-oxo-4-(²H₃)butanoic acid) enables tostereospecifically label with ¹³CDH₂ proS methyl groups of Valine (γ2methyl) and proS methyl groups of Leucine (δ2 methyl).

It can be used for ²H dynamic studies of proteins by liquid NMR.

The schema below shows how the amino acids Leucine and Valine arelabeled with this compound of formula 10.

The compound of formula 11(2-hydroxy-2-(²H₃)methyl-3-oxo-4-(²H,¹³C)butanoic acid) enables tostereospecifically label with ¹³CDH₂ proR methyl groups of Valine (γ1methyl) and proR methyl groups of Leucine (δ1 methyl) . . .

It can be used for ²H dynamic studies of proteins by liquid NMR. Thiscompound labels the amino acids Leucine and Valine in the manner shownin the following schema:

The compound of formula 12(2-(²H₅)ethyl-2-hydroxy-3-oxo-4-(²H,¹³C)butanoic acid) enables tospecifically label with ¹³CDH₂ γ2 methyl groups of Isoleucine.

It can be used for ²H dynamic studies of proteins by liquid NMR.

The compound of formula 12 label (L)-Isoleucine as shown in thefollowing schema:

The compound of formula 13(2-hydroxy-2-(²H₂,¹³C)methyl-3-oxo-4-(²H₃)butanoic acid) enables tostereospecifically label with ¹³CHD₂ proS methyl groups of Valine (γ2methyl) and proS methyl groups of Leucine (δ2 methyl).

It can be used for solid state NMR of proteins and dynamic studies ofproteins by liquid NMR.

The following schema shows how compound 13 labels Leucine and Valine inproteins:

The compound of formula 14(2-hydroxy-2-(²H₃)methyl-3-oxo-4-(²H₂,¹³C)butanoic acid) enables tostereospecifically label with ¹³CHD₂ proR methyl groups of Valine (γ1methyl) and proR methyl groups of Leucine (δ1 methyl).

It can be used for solid state NMR of proteins and for dynamic studiesof proteins by liquid NMR.

This compound labels Leucine and Valine as shown in the followingschema:

The compound of formula 15(2-(²H₅)ethyl-2-hydroxy-3-oxo-4-(²H₂,¹³C)butanoic acid) enables to labelwith ¹³CHD₂ specifically γ2 methyl groups of Isoleucine in proteins.

It can be used for solid state NMR of proteins and for dynamic studiesof proteins by liquid NMR.

This compound labels Isoleucine as shown in the following schema:

The compound of formula 16(U-(¹³C)-2-hydroxy-2-(²H₂)methyl-3-oxo-4-(²H₃)butanoic acid) can be usedfor solid state NMR of proteins.

It allows to label the carbon chain of Valine and Leucine and with¹³CHD₂ the proS methyl groups of Valine (γ2 methyl) and proS methylgroups of Leucine (δ2 methyl) in proteins as shown in the followingschema:

The compound of formula 17(U-(¹³C)-2-hydroxy-2-(²H₃)methyl-3-oxo-4-(²H₂)butanoic acid) can be usedfor solid state NMR of proteins.

It enables to label the carbon chain of Leucine and Valine and with¹³CHD₂ the proR methyl groups of Valine (γ1 methyl) and proR methylgroups of Leucine (δ1 methyl) in protein as shown in the followingschema:

The compound of formula 18(1,2,3,4-(¹³C)-2-(²H₅)ethyl-2-hydroxy-3-oxo-4-(²H₂)butanoic acid) can beused for solid state NMR of proteins

It enables to label the carbon chain and with ¹³CHD₂ the γ2 methylgroups of Isoleucine in proteins as shown in the following schema:

To summarize, among the acetolactate derivatives of formula I:

1) those more appropriate for assignment of NMR signals and/ormeasurements of structural restraints have the following formulae:

-   4=2-hydroxy-2-(¹³C)methyl-3-oxo-4(²H₃)butanoic acid,-   5=2-hydroxy-2-(²H₃)methyl-3-oxo-4(¹³C)butanoic acid,-   6=2-(²H₅)ethyl-2-hydroxy-3-oxo-4-(¹³C)methylbutanoic acid,-   9=1,2,3,4-(¹³C)-2-(²H₅)ethyl-2-hydroxy-3-oxobutanoic acid,-   21=1,2,3-(¹³C)-2-(¹³C)methyl-2-hydroxy-3-oxo-4-(²H₃)butanoic Acid,-   22=1,2,3,4-(¹³C)-2-(²H₃)methyl-2-hydroxy-3-oxobutanoic acid,-   24=1,2,    3-(¹³C)-2-(1′-(²H₂),¹³C₂)ethyl)-2-hydroxy-3-oxo-4-(²H₃)butanoic    acid,-   36=3,4-(¹³C)-2-(¹³C)methyl-2-hydroxy-3-oxo-4-(²H₃)butanoic acid,-   37=3,4-(¹³C)-2-(²H₃,¹³C)methyl-2-hydroxy-3-oxobutanoic acid-   40=3,4-(¹³C)-2-(¹³C)methyl-2-hydroxy-3-oxobutanoic acid,-   45=3,4-(¹³C)-2-(1′-(²H₂),¹³C₂)ethyl-2-hydroxy-3-oxo-4-(²H₃)butanoic    acid,-   46=3,4-(¹³C)-2-(²H₅,¹³C₂)ethyl-2-hydroxy-3-oxobutanoic acid,-   49=3,4-(¹³C)-2-(1′-(²H₂),¹³C₂)ethyl-2-hydroxy-3-oxobutanoic acid.

2) those which are preferably used for dynamics studies by liquid stateNMR and for use in solid state NMR are the compounds of the followingformulae:

-   13=2-hydroxy-2-(²H₂,¹³C)methyl-3-oxo-4-(²H₃)butanoic acid,-   14=2-hydroxy-2-(²H₃)methyl-3-oxo-4-(²H₂,¹³C)butanoic acid,-   15=2-(²H₅)ethyl-2-hydroxy-3-oxo-4-(²H₂,¹³C)butanoic acid,-   34=2-(1′-(²H₂),2′-(²H),2′-(¹³C))ethyl-2-hydroxy-3-oxo-4-(²H₃)butanoic    acid, and

3) for dynamics studies by ²H NMR, preferred acetolactate derivatives offormula I have the following formulae:

-   10=2-hydroxy-2-(²H,¹³C)methyl-3-oxo-4-(²H₃)butanoic acid,-   11=2-hydroxy-2-(²H₃)methyl-3-oxo-4-(²H,¹³C)butanoic acid,-   12=2-(²H₅)ethyl-2-hydroxy-3-oxo-4-(²H,¹³C)butanoic acid.

The labeling process of the invention was applied to malate synthase G(MSG), the largest monomeric protein (82 kDa) for which a 3 D structurehas been determined by NMR spectroscopy, using compound of formula 4.

The methyl-TROSY spectra of specifically protonated MSG over expressedin E. Coli in the presence of the compound 4 of the invention have beencompared with those of MSG over expressed in E. Coli in the presence of2-oxo-3-(²H₃)-3-(²H)-4-(¹³C)-ketoisovalerate.

A direct comparison of these spectra shows that replacingα-ketoisovalerate with compound of the invention increases thesensitivity of NMR spectra by a factor of 1.6 (theoretically 2) anddecreases the number of peaks by two.

Only signals for Val-γ₂ and Leu-δ₂ methyl groups can be observed whenthe protein is prepared using synthetic2-(¹³C)methyl-4-(²H₃)-acetolactate(compound of formula 4).

The absence of signals for Val-γ₁ and Leu-δ₁ methyl groups confirms thatthe ¹³C¹H₃ methyl substituent in position 2 of 2-(S)-acetolactate isstereospecifically transfered in-vivo to position proS of2,3-dihydroxy-isovalerate by ketol-isomerase and that no methylinterconversion occurs in the following steps of the Leu/Valbiosynthetic pathway.

Furthermore, no signals for CH₂D and CD₂H isotopomers were detecteddemonstrating that H/D exchange does not occur after the introduction ofacetolactate to M9/D₂O culture medium. Lastly, no scrambling to othermethine, methylene or methyl sites was detected, indicating that theexcess of acetolactate in-vivo does not interfere with other metabolicpathways.

Thus, the use of specifically-labeled acetolactate is an efficientmethod to realize a complete and stereospecific methyl-labeling of Leuand Val side chains in a fully perdeuderated protein background withoutdetectable scrambling.

As the acetolactate derivatives are not incorporated into othermetabolic pathways, they can be conveniently used in combination withother precursors such as those proposed for specific labeling ofIle-[CH₃]^(δ1), or Ala-[CH₃]^(β) methyl groups.

An obvious first application of this new labeling scheme is thestereospecific assignment of methyl groups of Leu, Val and Ile.

Despite the existence of efficient methods to connect methyl resonancesto sequentially-assigned backbone nuclei, the stereospecific assignmentof prochiral methyl groups remains difficult. Previous approaches haveinvolved either a fractional [¹³C]-labeling strategy or the measurementof small scalar couplings.

While these approaches have been shown to be useful for small and mediumsize proteins, their application to larger proteins is challenging. Incomparison, stereospecific assignment of Leu, Val, and Ile methyl groupscan be obtained directly by visually inspecting 2D methyl-TROSY spectrarecorded on specifically protonated proteins produced with compound offormula 4 according to the invention.

This strategy is directly applicable to large proteins as demonstratedfor MSG (82 kDa) for which 98% of Leu/Val methyl groups wereunambiguously assigned stereospecifically.

Specific protonation of methyl groups is a powerful method to extractlong-range distance restraints in proteins.

For large proteins the extraction of inter-methyl NOE is generallyhampered by the low intrinsic resolution of ¹³C-edited 4D NOESY spectra.

Simplification of spectra using the stereospecific labeling of prochiralmethyl groups according to the process of the invention significantlyreduces ambiguity in the analysis of Nuclear Over Hauser effect (NOE)cross peaks.

NOEs between proS and proR methyl groups are no longer detected usingstereospecifically-labeled samples. However, the use of the compound 4of the invention in place of2-oxo-3-(²H₃)-3-(²H)-4-(¹³C)-ketoisovalerate increases the protonationlevel of proS methyl groups theoretically two-fold, which in turnenhances the intensity of the remaining NOE cross-peaks by a factor oftheoretically 4.

This gain in sensitivity leads to the detection of new structurallymeaningful long-range NOE cross-peaks between more remote proS methylgroups, thereby compensating for the loss of NOEs involving proR methylgroups.

A comparison of distance restraints extracted from 4D NOESY spectrarevealed that stereospecific labeling of Leu/Val methyl groups increasedthe distance threshold for which an NOE can be detected by ˜20%.Prochiral-specific labeling of methyl groups is not only an attractiveway to simplify the time-consuming step of NOE analysis, it allows asignificant extension of the range of structurally meaningful ¹H-¹Hdistances that can be measured in large proteins.

The process of this invention can also be used for the specific isotopiclabeling of amino acids selected from the group consisting of Val, Leuand Ile, and more particularly for the specific labeling of methylgroups of Leu and Val as well as specific labeling of the γ2-methylgroups of isoleucine.

Another object of the invention is acetolactate derivatives of thefollowing formula I-1:

wherein:

-   -   X is an exchangeable hydrogen being ¹H or ²H (D) depending on        the nature of the solvent,    -   each Y is independently from the others ¹²C or ¹³C,    -   R¹ is a methyl group in which the carbon atom is ¹³C or ¹²C and        the hydrogen atoms are independently from each other ¹H or ²H        (D),    -   R² is either a methyl group in which the carbon atom is ¹³C or        ¹²C and the hydrogen atoms are independently from each other ¹H        or ²H (D), or an ethyl group in which the carbon atoms are        independently from each other ¹³C or ¹²C and the hydrogen atoms        are independently from each other ¹H or ²H (D),        at the provisos that:

1) the hydrogen atoms of the acetolactate derivative of Formula I arenot all, at the same time, either ¹H or ²H (D),

2) the hydrogen atoms of R¹ and the hydrogen atoms of R² are not all, atthe same time, either ¹H or ²H (D),

3) when all the hydrogen atoms of R¹ are ²H, then the carbon atoms, informula I-1, are not all, at the same time, ¹²C.

Preferred compounds of the invention have the formula I-1 in which:

-   -   R¹ is chosen among the following groups:    -   ¹²CH₃, ¹²CD₃, ¹³CH₃, ¹³CD₃, ¹³CHD₂, ¹³CH₂D,    -   R² is chosen among the following groups:    -   ¹²CH₃, ¹²CD₃, ¹³CH₃, ¹³CD₃, ¹³CHD₂, ¹³CH₂D, ¹²CH₃ ¹²CD₂, ¹²CD₃        ¹²CD₂, ¹³CH₃ ¹²CD₂, ¹³CH₃ ¹³CD₂, ¹³CD₃ ¹³CD₂, ¹³CHD₂ ¹³CD₂,        ¹³CH₂D¹³CD₂, ¹³CHD₂ ¹²CD₂, ¹³CH₂D¹²CD₂,    -   each Y is independently from the others ¹²C or ¹³C,        at the provisos that:

1) the hydrogen atoms of the acetolactate derivative of Formula I arenot all, at the same time, either ¹H or ²H (D),

2) the hydrogen atoms of R¹ and the hydrogen atoms of R² are not all, atthe same time, either ¹H or ²H (D), and

3) when all the hydrogen atoms of R¹ are ²H, then the carbon atoms, informula I-1 are not all, at the same time, ¹²C.

Preferred compounds of the invention are the compounds of followingformulae 2-57:

-   2=2-hydroxy-2-(²H₃)methyl-3-oxobutanoic acid,-   3=2-(²H₅)ethyl-2-hydroxy-3-oxobutanoic acid,-   4=2-hydroxy-2-(¹³C)methyl-3-oxo-4(²H₃)butanoic acid,-   5=2-hydroxy-2-(²H₃)methyl-3-oxo-4(¹³C)butanoic acid,-   6=2-(²H₅)ethyl-2-hydroxy-3-oxo-4-(¹³C)methylbutanoic acid,-   7=U-(¹³C)-2-hydroxy-2-methyl-3-oxo-4(²H₃)butanoic acid,-   8=U-(¹³C)-2-hydroxy-2-(²H₃)methyl-3-oxobutanoic acid,-   9=1,2,3,4-(¹³C)-2-(²H₅)ethyl-2-hydroxy-3-oxobutanoic acid,-   10=2-hydroxy-2-(²H,¹³C)methyl-3-oxo-4-(²H₃)butanoic acid,-   11=2-hydroxy-2-(²H₃)methyl-3-oxo-4-(²H,¹³C)butanoic acid,-   12=2-(²H₅)ethyl-2-hydroxy-3-oxo-4-(²H,¹³C)butanoic acid,-   13=2-hydroxy-2-(²H₂,¹³C)methyl-3-oxo-4-(²H₃)butanoic acid,-   14=2-hydroxy-2-(²H₃)methyl-3-oxo-4-(²H₂,¹³C)butanoic acid,-   15=2-(²H₅)ethyl-2-hydroxy-3-oxo-4-(²H₂,¹³C)butanoic acid,-   16=U-(¹³C)-2-hydroxy-2-(²H₂)methyl-3-oxo-4-(²H₃)butanoic acid,-   17=U-(¹³C)-2-hydroxy-2-(²H₃)methyl-3-oxo-4-(²H₂)butanoic acid,-   18=1,2,3,4-(¹³C)-2-(²H₅)ethyl-2-hydroxy-3-oxo-4-(²H₂)butanoic acid,-   19=2-(1′-(²H₂)ethyl-2-hydroxy-3-oxo-4-(²H₃)butanoic acid,-   20=2-((1′-(²H₂),2′-(¹³C))ethyl-2-hydroxy-3-oxo-4-(²H₃)butanoic acid,-   21=1,2,3-(¹³C)-2-(¹³C)methyl-2-hydroxy-3-oxo-4-(²H₃)butanoic acid,-   22=1,2,3,4-(¹³C)-2-(²H₃)methyl-2-hydroxy-3-oxobutanoic acid,-   23=U-(¹³C)-2-(²H₅)ethyl-2-hydroxy-3-oxobutanoic acid,-   24=1,2,3-(¹³C)-2-(1′-(²H₂),¹³C₂)ethyl)-2-hydroxy-3-oxo-4-(²H₃)butanoic    acid,-   25=U-(¹³C)-2-(1′-(²H₂))ethyl-2-hydroxy-3-oxo-4-(²H₃)butanoic acid,-   26=U-(¹³C)-2-(²H)methyl-2-hydroxy-3-oxo-4-(²H₃)butanoic acid,-   27=U-(¹³C)-2-(²H₃)methyl-2-hydroxy-3-oxo-4-(²H)butanoic acid,-   28=U-(¹³C)-2-((²H₅)ethyl)-2-hydroxy-3-oxo-4-(²H)butanoic acid,-   29=1,2,3-(¹³C)-2-(²H,¹³C)methyl-2-hydroxy-3-oxo-4-(²H₃)butanoic    acid,-   30=1,2,3,4-(¹³C)-2-(²H₃)methyl-2-hydroxy-3-oxo-4-(²H)butanoic acid,-   31=1,2,3,4-(¹³C)-2-(²H₅)ethyl-2-hydroxy-3-oxo-4-(²1H)butanoic acid,-   32=U-(¹³C)-2-(1′-(²H₂),2′-(²H))ethyl-2-hydroxy-3-oxo-4-(²H₃)butanoic    acid,-   33=1,2,3-(¹³C)-2-(2′-(²H),¹³C₂)ethyl-2-hydroxy-3-oxo-4-(²H₃)butanoic    acid,-   34=2-(1′-(²H₂),2′-(²H),2′-(¹³C))ethyl-2-hydroxy-3-oxo-4-(²H₃)butanoic    acid,-   35=1,2,3-(¹³C)-2-(²H₂,¹³C)methyl-2-hydroxy-3-oxo-4-(²H₃)butanoic    acid,-   36=3,4-(¹³C)-2-(¹³C)methyl-2-hydroxy-3-oxo-4-(²H₃)butanoic acid,-   37=3,4-(¹³C)-2-(²H₃,¹³C)methyl-2-hydroxy-3-oxobutanoic acid,-   38=3,4-(¹³C)-2-(²H₂,¹³C)methyl-2-hydroxy-3-oxo-4-(²H₃)butanoic acid,-   39=3,4-(¹³C)-2-(²H,¹³C)methyl-2-hydroxy-3-oxo-4-(²H₃)butanoic acid,-   40=3,4-(¹³C)-2-(¹³C)methyl-2-hydroxy-3-oxobutanoic acid,-   41=3,4-(¹³C)-2-(²H,¹³C)methyl-2-hydroxy-3-oxo-4-(²H)butanoic acid,-   42=3,4-(¹³C)-2-(²H₂,¹³C)methyl-2-hydroxy-3-oxo-4-(²H₂)butanoic acid,-   43=3,4-(¹³C)-2-(²H₃,¹³C)methyl-2-hydroxy-3-oxo-4-(²H₂)butanoic acid,-   44=3,4-(¹³C)-2-(²H₃,¹³C)methyl-2-hydroxy-3-oxo-4-(²H)butanoic acid,-   45=3,4-(¹³C)-2-(1′-(²H₂),¹³C₂)ethyl-2-hydroxy-3-oxo-4-(²H₃)butanoic    acid,-   46=3,4-(¹³C)-2-(²H₅,¹³C₂)ethyl-2-hydroxy-3-oxobutanoic acid,-   47=3,4-(¹³C)-2-(1′-(²H₂),2′-(²H),¹³C₂)ethyl-2-hydroxy-3-oxo-4-(²H₃)butanoic    acid,-   48=3,4-(¹³C)-2-(1′-(²H₂),2′-(²H₂),¹³C₂)ethyl-2-hydroxy-3-oxo-4-(²H₃)butanoic    acid,-   49=3,4-(¹³C)-2-(1′-(²H₂),¹³C₂)ethyl-2-hydroxy-3-oxobutanoic acid,-   50=3,4-(¹³C)-2-(1′-(²H₂),2′-(²H₂),¹³C₂)ethyl-2-hydroxy-3-oxo-4-(²H₂)butanoic    acid,-   51=3,4-(¹³C)-2-(1′-(²H₂)-2′-(²H),¹³C₂)ethyl-2-hydroxy-3-oxo-4-(²H)butanoic    acid,-   52=3,4-(¹³C)-2-(2H₅,¹³C₂)ethyl-2-hydroxy-3-oxo-4-(²H₂)butanoic acid,-   53=3,4-(¹³C)-2-(2H₅,¹³C₂)ethyl-2-hydroxy-3-oxo-4-(²H)butanoic acid,-   54=U-(¹³C)-2-(²H₅)ethyl-2-hydroxy-3-oxo-4-(²H₂)butanoic acid,-   55=U-(¹³C)-2-(²H₅)ethyl-2-hydroxy-3-oxo-4-(²H)butanoic acid,-   56=1,2,3-(¹³C)-2-(1′-(²H₂),2′-(²H₂),¹³C₂)ethyl-2-hydroxy-3-oxo-4-(²H₃)butanoic    acid,-   57=U-(¹³C)-2-(1′-(²H₂),2′-(²H₂))ethyl-2-hydroxy-3-oxo-4-(²H₃)butanoic    acid.

Among these compounds, the compounds of the following formulae arepreferred:

-   4=2-hydroxy-2-(¹³C)methyl-3-oxo-4(²H₃)butanoic acid,-   5=2-hydroxy-2-(²H₃)methyl-3-oxo-4(¹³C)butanoic acid,-   6=2-(²H₅)ethyl-2-hydroxy-3-oxo-4-(¹³C)methylbutanoic acid,-   9=1,2,3,4-(¹³C)-2-(²H₅)ethyl-2-hydroxy-3-oxobutanoic acid,-   21=1,2,3-(¹³C)-2-(¹³C)methyl-2-hydroxy-3-oxo-4-(²H₃)butanoic Acid,-   22=1,2,3,4-(¹³C)-2-(²H₃)methyl-2-hydroxy-3-oxobutanoic acid,-   24=1,2,3-(¹³C)-2-(1′-(²H₂),¹³C₂)ethyl)-2-hydroxy-3-oxo-4-(²H₃)butanoic    acid,-   36=3,4-(¹³C)-2-(¹³C)methyl-2-hydroxy-3-oxo-4-(²H₃)butanoic acid,-   37=3,4-(¹³C)-2-(²H₃,¹³C)methyl-2-hydroxy-3-oxobutanoic acid-   40=3,4-(¹³C)-2-(¹³C)methyl-2-hydroxy-3-oxobutanoic acid,-   45=3,4-(¹³C)-2-(1′-(²H₂),¹³ C₂)ethyl-2    -hydroxy-3-oxo-4-(²H₃)butanoic acid,-   46=3,4-(¹³C)-2-(²H₅,¹³C₂)ethyl-2-hydroxy-3-oxobutanoic acid,-   49=3,4-(¹³C)-2-(1′-(²H₂),¹³C₂)ethyl-2-hydroxy-3-oxobutanoic acid.

But still other preferred compounds have the following formulae:

-   13=2-hydroxy-2-(²H₂,¹³C)methyl-3-oxo-4-(²H₃)butanoic acid,-   14=2-hydroxy-2-(²H₃)methyl-3-oxo-4-(²H₂,¹³C)butanoic acid,-   15=2-(²H₅)ethyl-2-hydroxy-3-oxo-4-(²H₂,¹³C)butanoic acid,-   34=2-(1′-(²H₂),2′-(²H),2′-(¹³C))ethyl-2-hydroxy-3-oxo-4-(²H₃)butanoic    acid.

Other preferred acetolactate derivatives according to the invention havethe following formulae:

-   10=2-hydroxy-2-(²H,¹³C)methyl-3-oxo-4-(²H₃)butanoic acid,-   11=2-hydroxy-2-(²H₃)methyl-3-oxo-4-(²H,¹³C)butanoic acid,-   12=2-(²H₅)ethyl-2-hydroxy-3-oxo-4-(²H,¹³C)butanoic acid.

The invention also proposes a process for manufacturing a compound offormula I, more particularly of formula 1-18, which comprises thefollowing steps:

a) alkylation, with a methyl group in which the carbon atom is ¹³C or¹²C and the hydrogen atoms are independently from each other ¹H or ²H(D), or an ethyl group in which the carbon atoms are independently fromeach other ¹³C or ¹²C and the hydrogen atoms are independently from eachother ¹H or ²H (D), of 3-oxobutanoate having its hydroxyl group inposition 1 protected by a protecting group, preferably a methyl or ethylgroup,

b) hydroxylation of the compound obtained in step a),

c) optionally deprotection and exchange of the desired ¹H atoms by ²Hatoms, wherein step b) of hydroxylation is carried out by usingdimethyldioxirane in presence of Nickel (II) ions.

Step b) of hydroxylation according to the process of the invention,enables to improve the total yield of the process to more than 90%whereas when this hydroxydation step is carried out using molecularoxygen in presence of Cobalt or Cerium ions as a catalyst, as suggestedby the prior art, the total yield of the process is only of about 50%without any other steps are modified.

The labeling process of the invention is particularly appropriate foranalysing proteins, including those included in biomolecular assembliesby NMR.

Thus, the invention also proposes processes for analysing proteins byNMR.

In a first embodiment, this process comprises a step of labeling ofproteins to be analysed by the process of labeling of the invention.

In a second embodiment, this process comprises a step of labeling ofproteins to be analysed with a compound if the following formula I:

wherein:

-   -   X is ¹H or ²H (D),    -   each Y is independently from the others ¹²C or ¹³C,    -   R¹ is a methyl group in which the carbon atom is ¹³C or ¹²C and        the hydrogen atoms are independently from each other ¹H or ²H        (D),    -   R² is either a methyl group in which the carbon atom is ¹³C or        ¹²C and the hydrogen atoms are independently from each other ¹H        or ²H (D), or an ethyl group in which the carbon atoms are        independently from each other ¹³C or ¹²C and the hydrogen atoms        are independently from each other ¹H or ²H (D),        at the provisos that:

1) the hydrogen atoms of the acetolactate derivative of Formula I arenot all, at the same time, either ¹H or ²H (D),

2) the hydrogen atoms of R¹ and the hydrogen atoms of R² are not all, atthe same time, either ¹H or ²H (D).

In a third embodiment, this process comprises a step of labeling ofproteins to be analysed with a compound if the following formula I-1:

wherein:

-   -   X is ¹H or ²H (D),    -   each Y is independently from the others ¹²C or ¹³C,    -   R¹ is a methyl group in which the carbon atom is ¹³C or ¹²C and        the hydrogen atoms are independently from each other ¹H or ²H        (D),    -   R² is either a methyl group in which the carbon atom is ¹³C or        ¹²C and the hydrogen atoms are independently from each other ¹H        or ²H (D), or an ethyl group in which the carbon atoms are        independently from each other ¹³ C or ¹²C and the hydrogen atoms        are independently from each other ¹H or ²H (D),        at the provisos that:

1) the hydrogen atoms of the acetolactate derivative of Formula I arenot all, at the same time, either ¹H or ²H (D),

2) the hydrogen atoms of R¹ and the hydrogen atoms of R² are not all, atthe same time, either ¹H or ²H (D),

3) when all the hydrogen atoms of R¹ are ²H, then the carbon atoms, informula I-1, are not all, at the same time, ¹²C.

In order that the invention be better understood, an example of carryingout the labeling process of the invention is given below. This exampleis only illustrative, and in no way limitative of the invention.

1. Synthesis of Selectively Methyl-Labeled Acetolactate

Ethyl 2-(¹³C)methyl-3-oxobutanoate

A mixture of 12.15 mL (95.4 mmol) of ethyl 3-oxobutanoate (A), 14.5 g(104.9 mmol) K₂CO₃ and 15.0 g (104.9 mmol) ¹³C-methyl iodide (CambridgeIsotope Laboratories, Inc.) in 120 mL of absolute ethanol was heated at40° C. under argon for 90 h. After filtration, the filtrate wasconcentrated in vacuo to afford 12.30 g (yield 89%) of product, whichwas sufficiently pure to be used without further purification. ¹H NMR(CDCl₃); 4.21 (q, J=7.1 Hz, 2H), 3.51 (dq, J=7.2, 4.4 Hz, 1H), 2.25 (s,3H), 1.35(dd, J=130.4, 7.2 Hz, 3H), 1.29 (t, J=7.1 Hz, 3H).

Ethyl 2-hydroxy-2-(¹³C)methyl-3-oxobutanoate (B)

Hydroxylation reaction was carried out by freshly prepareddimethyldioxirane in presence of Nickel(II) ions. To a mixture of 50 mg(0.345 mmol) of ethyl 2-(¹³C)methyl-3-oxobutanoate in 3 mL of distilledwater, was added successively 8.6 mg (0.035 mmole) of Ni(OAc)₂.4H₂O and,at 0° C., 20 mL of an untitrated solution of dimethyldioxirane(0.05-0.10 M) in acetone. The resulting solution was allowed to warm toroom temperature and stirred for 24 hours. The organic solvant was thenevaporated in vacuo and the resulting aqueous residue was extracted withdichloromethane (four times). The organic extract was dried over Na₂SO₄and concentrated in vacuo to afford 51 mg (0.317 mmol; 92% yield; >90%conversion) of ethyl 2-hydroxy-2-(¹³C)methyl-3-oxobutanoate as acolorless liquid which was pure enough to be used without furtherpurification.

NMR spectroscopy: ¹H NMR (CDCl₃); 4.25(q, J=7.1 Hz, 2H), 2.27 (s, 3H),1.58 (d, ¹J_(H-13C)=130.3 Hz, 3H), 1.29 (t, J=7.1 Hz, 3H).2-hydroxy-2-(¹³C)methyl-3-oxo-4-(²H₃)-butanoate (or2-(¹³C)methyl-4-(²H₃)-acetolactate) (Compound of Formula 4)

Deprotection and exchange of the protons of the methyl group in position4 of ethyl 2-hydroxy-2-(¹³C)methyl-3-oxobutanoate (B) were achieved inD₂O at pH 13. Typically, 300 mg of B was added to 24 mL of a 0.1 MNaOD/D₂O solution. The deprotection was immediate as observed by NMRspectroscopy. The completion of the exchange on the 4-methyl was alsofollowed in real time by NMR spectrometry. 97±1% of protons of terminalmethyl groups have been exchanged after 30 min while the methylsubsistent in position 2 remains protonated at a level of 98±1%. As thedeprotection reaction consumes hydroxide ions, the pH and consequentlythe deuterium exchange rate decreases during the reaction. Once theexchange was complete, the solution was adjusted to neutral pH with DCland 2 mL of 1 M TRIS pH 8 in D₂O was added. The solution of the compoundof formula 4 was then stored at −20° C. until required.

Reaction scheme of the protocol for the production of U-[²H], U-[¹⁵N],Leu/Val-[¹³C¹H₃]^(proS) labeled proteins.

Detailed protocol for the chemical synthesis of2-(¹³C)methyl-4-(²H₃)-acetolactate is presented above. ¹³C nuclei aredisplayed in italic bold. The stereochemistry, following theincorporation of ¹³C¹H₃ group into acetolactate, in the differentintermediates of Leu/Val biogenesis pathway is indicated on the figure(assuming growth in M9/D₂O based culture medium). Each biosyntheticintermediate has been named according to the Kyoto Encyclopedia of Genesand Genomes. The enzymes responsible for catalyzing reaction areindicated by EC number. EC 1.1.1.86: ketol-acid reductoisomerase; EC4.2.1.9: dihydroxy-acid dehydratase; EC 2.6.1.42: branched-chain aminoacid aminotransferase; EC 2.3.3.13: 2-isopropylmalate synthase; EC4.2.1.33: 3-isopropylmalate dehydratase; EC 1.1.1.85: 3-isopropylmalatedehydrogenase. Further information on the Leu/Val metabolic pathway canbe found online: http://www.genome.jp/kegg/.

2. Overexpression of methyl stereospecifically labeled proteins in E.coli.Optimization of the incorporation of acetolactate in overexpressedprotein.

Initial experiments to determine the level of acetolactate incorporationinto overexpressed proteins were performed using ubiquitin as a modelsystem. E. coli BL21(DE3) cells were transformed with a pET41c plasmidcarrying the human His-tagged ubiquitin (pET41c-His-Ubi) gene andtransformants were grown in M9/D₂O media containing 1 g/L ¹⁵ND₄Cl, and 2g/L of U-[²H], U-[¹³C], glucose. When the optical density (O.D.) at 600nm reached 0.8, a solution containing unlabeled acetolactate was added.After an additional 1 h, protein expression was induced by the additionof IPTG to a final concentration of 1 mM. Induction was performed for 3hours at 37° C. Ubiquitin was purified by Ni-NTA (Qiagen) chromatographyin a single step.

The optimal quantity of acetolactate required to achieve near completeincorporation in the overexpressed protein was assessed in a series ofcultures (90 mL each) in which different amounts of unlabeled precursorwere added 1 hour prior induction to final concentrations of 0, 100,200, 300 and 800 mg/L. The level of incorporation into the purifiedprotein was monitored by directly-detected ¹³C 1D NMR. When theprecursor is incorporated into the overexpressed protein, the ¹³C-L,Vresidues are replaced by amino acids with ¹²C side chains. Thequantification was performed by comparing the integral of signals of 4isolated Leu/Val methyl resonances (19-21 ppm) with respect to thesignals of the methyl groups of Ile, Ala (between 9-19 ppm). Theaddition of 300 mg of pure acetolactate per liter of M9/D₂O culturemedium achieves an incorporation level of 95% in Leu/Val side chainswithout detectable scrambling to other amino-acid biogenesis pathways

Production of U-[²H], U-[¹⁵N], Leu/Val-[¹³C¹H₃]^(proS) proteins.

E. coli BL21(DE3) carrying the plasmid of the overexpressed protein(TET2 or MSG) were progressively adapted, in three stages, over 24 h, toM9/D₂O media containing 1 g/L ¹⁵ND₄Cl and 2 g/L D-glucose-d₇ (Isotec).In the final culture, the bacteria were grown at 37° C. in M9 mediaprepared with 99.85% D₂O (Eurisotop). When the O.D. (600 nm) reached0.8, a solution containing 2-(¹³C)methyl-4-(²H₃)-acetolactate (compoundof formula 4) (prepared with the protocol described above) was added.Acetolactate was added to the culture medium to a final concentration of300 mg/L. 1 hour later, TET2 (/MSG) expression was induced by theaddition of IPTG to a final concentration of 1 mM (/0.1 mM). Expressionwas performed for 3 hours (/12 hours) at 37° C. (/20° C.) beforeharvesting. For MSG, ¹³C spectra were recorded at 37° C. in D₂O on a NMRspectrometer operating at a proton frequency of 600 MHz. Only signalsfor Leu and Val methyl carbons were observed in ¹³C spectra, indicatingthat ¹³C¹H₃ groups of acetolactate were not incorporated in metabolicpathway of other amino-acids.

Production of U-[²H], U-[¹⁵N], Ile-[¹³C¹H₃]^(γ2) proteins.

E. coli BL21(DE3) carrying the plasmid of the overexpressed protein(TET2 or MSG) were progressively adapted, in three stages, over 24 h, toM9/D₂O media containing 1 g/L ¹⁵ND₄Cl and 2 g/L D-glucose-d₇ (Isotec).In the final culture, the bacteria were grown at 37° C. in M9 mediaprepared with 99.85% D₂O (Eurisotop). When the O.D. (600 nm) reached0.8, a solution containing 2-(²H₅)ethyl-2-hydroxy-3-oxo-4(¹³C)butanoate(compound of formula 6) (prepared with the protocol described above) wasadded. Product was added to the culture medium to a final concentrationof 300 mg/L. 1 hour later, TET2 (/MSG) expression was induced by theaddition of IPTG to a final concentration of 1 mM (/0.1 mM). Expressionwas performed for 3 hours (/12 hours) at 37° C. (/20° C.) beforeharvesting.

Production of U-[²H], U-[¹⁵N], Leu/Val-[¹³C¹H₃, ¹²C²H₃] proteins.

For aim of comparison, the production of perdeuterated proteins withnon-stereospecific ¹³C¹H labeling of Leu and Val methyl groups wasachieved using the protocol used before the invention, i.e. the protocoldescribed by V. Tugarinov et al., J. Biomol. NMR 2004, 28, 165-172 andR. Lichtenecker et al., J. Am. Chem. Soc. 2004, 126, 5348-5349.

This protocol is the protocol described above but with the addition 1hour prior induction of 125 mg/L of3-(²H₃)methyl-3-(²H)-4-(¹³C)-ketoisovalerate (Isotec) in place of 300mg/L of labeled acetolactate (compound of formula 4).

Production of U-[²H], U-[¹⁵N], U-[¹²C], U-[¹³C¹H₃]^(proS)-Leu/Val,U-[¹³C¹H₃]-Ala proteins.

The production of perdeuterated proteins with stereospecific ¹³C¹Hlabeling of Leu/Val proS methyl groups and Ala-positions was achievedusing the general protocol described above but with the addition 1 hourprior induction of 800 mg/L of 2-(S)-2-(²H)-3-(¹³C)-Alanine (CortecNet)together with 300 mg/L of labeled acetolactate (compound of formula 4).A 2D ¹³C-methyl TROSY spectra was recorded at 37° C. in D₂O on a NMRspectrometer operating at a proton frequency of 800 MHz. Only peakscorresponding to the expected signals of Alanine methyl groups arid proSmethyl groups of Leu and Val side chains were observed indicating thatlabeling using acetolactate derivatives does not interference with othermethyl labeling processes.

Proteins Purification.

Malate Synthase G (MSG) was purified initially by Chelating Sepharosechromatography (GE Healthcare) followed by gel filtration chromatography(Superdex 200 pg GE Healthcare). Typical final yields after purificationwere 60-80 mg/L of methyl specific protonated MSG. The concentration ofMSG in typical NMR samples was 1 mM in 100% D₂O buffer containing 25 mMMES (pH 7.0 uncorrected), 20mM MgCl₂, 5 mM DTT. NMR data were acquiredat 37° C.

TET2 was purified using two anion exchange chromatography steps (DEAESepharose CL-6B, and Resource Q 6 mL, GE Healthcare) followed by gelfiltration (Sephacryl S-300 HR, GE Healthcare). Typical final yieldafter purification was 20 mg/L of methyl specific protonated TET2.Samples prepared in this manner were demonstrated to be fully active(measured by hydrolytic activity using Leu-4-nitroanitide). The finalNMR samples of TET consisted of ˜80 μM TET2 dodecamer (˜1 mM monomer) in20 mM Tris (pH 7.4 uncorrected), 20 mM NaCl dissolved in 300 μL D₂O. NMRdata were acquired at 50° C.

3. NMR Spectroscopy. Experimental Details.

All ¹H and ¹³C 1D NMR spectra of ubiquitin and MSG were recorded on aVarian DirectDrive spectrometer operating at a proton frequency of 600MHz equipped with a cryogenic triple resonance pulsed field gradientprobehead.

2D methyl-TROSY spectra were recorded on a Varian DirectDrivespectrometer operating at a proton frequency of 800 MHz equipped with acryogenic triple resonance pulsed field gradient probehead. The ¹H-¹³CHMQC of MSG (/TET2) were recorded with 1288 (/780) complex data pointsin direct dimension (maximum t₂=99 ms (/60 ms)) and 512 (/380) points incarbon dimension (maximum t₁=128 ms (/47 ms)).

The 4D HMQC-NOESY-HMQC experiments were recorded on a Varian DirectDrivespectrometer operating at a proton frequency of 800 MHz equipped with acryogenic triple resonance pulsed field gradient probehead. Data wereacquired in 96 h of a 1 mM sample of MSG with a NOE mixing time of 300ms. The experiments were collected with 20 complex points in theindirect ¹H dimension (maximum t₁=30 ms), 36 and 18 complex points inthe first and second carbon dimension (maximum t₂=21 ms & t₃=11 ms), and201 complex points in the direct dimension (maximum t₄=80 ins) and 4scans per increment. All data were processed and analyzed usingnmrPipe/nmrDraw and NMRView. Distances were quantified using a fullrelaxation matrix analysis of NOEs between remote protons inmethyl-specific protonated proteins as described in Sounier et al., J.Am. Chem. Soc. 2007, 129, 472-473.

Comparison of methyl-TROSY spectra recorded on Leu/Valmethyl-specifically labeled TET2 samples (468 kDa).

2D ¹³C-methyl TROSY spectra were recorded at 50° C. in D₂O on a NMRspectrometer operating at a proton frequency of 800 MHz for U-[²H],U-[¹²C], Leu/Val-[¹²C²H₃, ¹³C¹H₃] TET2 with non-stereospecific[¹³C¹H₃]-methyl labeling prepared using3-(²H₃)methyl-3-(²H)-4-(¹³C)-ketoisovalerate; and for U-[²H], U-[¹²C],Leu/Val-[¹³C¹H₃]^(proS) TET2 with stereospecific labeling prepared using2-(13C)methyl-4-(²H₃)-acetolactate (compound of formula 4). Preparationof TET2 assembly using a non-stereoselectively labeling scheme resultsin spectra with substantial cross-peak overlap that would have greatlyhampered the observation of amastatin-induced chemical shift changes(Amastatin is an inhibitor of TET2).

1. A process for the specific isotopic labeling of at least one aminoacid selected from the group consisting of Valine (Val), Leucine (Leu),and Isoleucine (Ile), in a protein or a biomolecular assembly, theprocess comprising a): introducing, in a medium comprising bacteriaoverexpressing a protein, an acetolactate compound having a formula I:

wherein: X is ¹H or ²H (D), each Y is independently ¹²C or ¹³C, R¹ is amethyl group whose carbon atom is ¹³C or ¹²C and whose hydrogen atomsare each independently ¹H or ²H (D), R² is (i) a methyl group whosecarbon atom is ¹³C or ¹²C and whose hydrogen atoms are eachindependently ¹H or ²H (D), or (ii) an ethyl group whose carbon atomsare each independently ¹³C or ¹²C and whose hydrogen atoms are eachindependently ¹H or ²H (D), with the provisos that: 1) the hydrogenatoms of the acetolactate compound are not all, at the same time, either¹H or ²H (D), and 2) the hydrogen atoms of R¹ and the hydrogen atoms ofR² are not all, at the same time, either ¹H or ²H (D).
 2. The process ofclaim 1 further comprising: b) overexpression of the protein by thebacteria in the medium, and c) purification of the protein.
 3. Theprocess of claim 1 wherein: R¹ is selected from the group consisting of:¹²CH₃, ¹²CD₃, ¹³CH₃, ¹³CD₃, ¹³CHD₂, and ¹³CH₂D, R²is selected from thegroup consisting of: ¹²CH₃, ¹²CD₃, ¹³CH₃, ¹³CD₃, ¹³CHD₂, ¹³CH₂D, ¹²CH₃¹²CD₂, ¹²CD₃ ¹²CD₂, ¹³CH₃ ¹²CD₂, ¹³CH₃ ¹³CD₂, ¹³CD₃ ¹³CD₂, ¹³CHD₂ ¹³CD₂,¹³CH₂D¹³CD₂, ¹³CHD₂ ¹²CD₂, and ¹³CH₂D¹²CD₂.
 4. The process of claim 1,wherein the acetolactate compound is selected from the group consistingof: 2-hydroxy-2-(¹³C)methyl-3-oxo-4(²H₃)butanoic acid (formula 4),2-hydroxy-2-(²H₃)methyl-3-oxo-4(¹³C)butanoic acid (formula 5),2-(²H₅)ethyl-2-hydroxy-3-oxo-4-(¹³C)methylbutanoic acid (formula 6),1,2,3,4-(¹³C)-2-(²H₅)ethyl-2-hydroxy-3-oxobutanoic acid (formula 9),1,2,3-(¹³C)-2-(¹³C)methyl-2-hydroxy-3-oxo-4-(²H₃)butanoic acid (formula21), 1,2,3,4-(¹³C)-2-(²H₃)methyl-2-hydroxy-3-oxobutanoic acid (formula22), 1,2,3-(¹³C)-2-(1′-(²H₂),¹³C₂)ethyl)-2-hydroxy-3-oxo-4-(²H₃)butanoicacid (formula 24),3,4-(13C)-2-(13C)methyl-2-hydroxy-3-oxo-4-(2H3)butanoic acid (formula36), 3,4-(13C)-2-(2H3,13C)methyl-2-hydroxy-3-oxobutanoic acid (formula37), 3,4-(¹³C)-2-(¹³C)methyl-2-hydroxy-3-oxobutanoic acid (formula 40),3,4-(¹³C)-2-(1′-(²H₂),¹³C₂)ethyl-2-hydroxy-3-oxo-4-(²H₃)butanoic acid(formula 45), 3,4-(¹³C)-2-(²H₅,¹³C₂)ethyl-2-hydroxy-3-oxobutanoic acid(formula 46), and3,4-(¹³C)-2-(1′-(²H₂),¹³C₂)ethyl-2-hydroxy-3-oxobutanoic acid (formula49).
 5. The process of claim 1, wherein the acetolactate compound isselected from the group consisting of:2-hydroxy-2-(²H₂,¹³C)methyl-3-oxo-4-(²H₃)butanoic acid (formula 13),2-hydroxy-2-(²H₃)methyl-3-oxo-4-(²H₂,¹³C)butanoic acid (formula 14),2-(²H₅)ethyl-2-hydroxy-3-oxo-4-(²H₂,¹³C)butanoic acid (formula 15), and2-(1′-(²H₂),2′-(²H),2′-(¹³C))ethyl-2-hydroxy-3-oxo-4-(²H₃)butanoic acid(formula 34).
 6. The process of claim 1, wherein the acetolactatecompound is selected from the group consisting of:2-hydroxy-2-(²H,¹³C)methyl-3-oxo-4-(²H₃)butanoic acid (formula 10),2-hydroxy-2-(²H₃)methyl-3-oxo-4-(²H,¹³C)butanoic acid (formula 11), and2-(²H₅)ethyl-2-hydroxy-3-oxo-4-(²H,¹³C)butanoic acid (formula 12).
 7. Acompound having a formula I-1:

wherein: X is ¹H or ²H (D), each Y is independently ¹²C or ¹³C, R¹ is amethyl group whose carbon atom is ¹³C or ¹²C and whose hydrogen atomsare each independently ¹H or ²14 (D), R² is (i) a methyl group whosecarbon atom is ¹³C or ¹²C and whose hydrogen atoms are eachindependently ¹H or ²H (D), or (ii) an ethyl group whose carbon atomsare each independently ¹³C or ¹²C and whose hydrogen atoms are eachindependently ¹H or ²H (D), with the provisos that: 1) the hydrogenatoms of the compound are not all, at the same time, either ¹H or ²H(D), 2) the hydrogen atoms of R¹ and the hydrogen atoms of R² are notall, at the same time, either ¹H or ²H (D), and 3) when all the hydrogenatoms of R¹ are ²H, then the carbon atoms, in formula I-1, are not all,at the same time, ¹²C.
 8. The compound of claim 7, wherein: R¹ isselected from the group consisting of: ¹²CH₃, ¹²CD₃, ¹³CH₃, ¹³CD₃,¹³CHD₂, and ¹³CH₂D, R² is selected from the group consisting of: ¹²CH₃,¹²CD₃, ¹³CH₃, ¹³CD₃, ¹³CHD₂, ¹³CH₂D, ¹²CH₃ ¹²CD₂, ¹²CD₃ ¹²CD₂, ¹³CH₃¹²CD₂, ¹³CH₃ ¹³CD₂, ¹³CD₃ ¹³CD₂, ¹³CHD₂ ¹³CD₂, ¹³CH₂D¹³CD₂, ¹³CHD₂¹²CD₂, and ¹³CH₂D¹²CD₂.
 9. The compound of claim 7, which is selectedfrom the group consisting of:2-hydroxy-2-(¹³C)methyl-3-oxo-4(²H₃)butanoic acid (formula 4),2-hydroxy-2-(²H₃)methyl-3-oxo-4(¹³C)butanoic acid (formula 5),2-(²H₅)ethyl-2-hydroxy-3-oxo-4-(¹³C)methylbutanoic acid (formula 6),1,2,3,4-(¹³C)-2-(²H₅)ethyl-2-hydroxy-3-oxobutanoic acid (formula 9),1,2,3-(¹³C)-2-(¹³C)methyl-2-hydroxy-3-oxo-4-(²H₃)butanoic acid (formula21), 1,2,3,4-(¹³C)-2-(²H₃)methyl-2-hydroxy-3-oxobutanoic acid (formula22), 1,2,3-(¹³C)-2-(1′-(²H₂),¹³C₂)ethyl)-2-hydroxy-3-oxo-4-(²H₃)butanoicacid (formula 24),3,4-(¹³C)-2-(¹³C)methyl-2-hydroxy-3-oxo-4-(²H₃)butanoic acid (formula36), 3,4-(¹³C)-2-(²H₃,³C)methyl-2-hydroxy-3-oxobutanoic acid (formula37), 3,4-(¹³C)-2-(¹³C)methyl-2-hydroxy-3-oxobutanoic acid (formula 40),3,4-(¹³C)-2-(1′-(²H₂),¹³C₂)ethyl-2-hydroxy-3-oxo-4-(²H₃)butanoic acid(formula 45), 3,4-(¹³C)-2-(²H₅,¹³C₂)ethyl-2-hydroxy-3-oxobutanoic acid(formula 46), and3,4-(¹³C)-2-(1′-(²H₂),¹³C₂)ethyl-2-hydroxy-3-oxobutanoic acid (formula49).
 10. The compound of claim 7, which is selected from the groupconsisting of: 2-hydroxy-2-(²H₂,¹³C)methyl-3-oxo-4-(²H₃)butanoic acid(formula 13), 2-hydroxy-2-(²H₃)methyl-3-oxo-4-(²H₂,¹³C)butanoic acid(formula 14), 2-(²H₅)ethyl-2-hydroxy-3-oxo-4-(²H₂,¹³C)butanoic acid(formula 15), and2-(1′-(²H₂),2′-(²H),2′-(¹³C))ethyl-2-hydroxy-3-oxo-4-(²H₃)butanoic acid(formula 34).
 11. The compound of claim 7, which is selected from thegroup consisting of: 2-hydroxy-2-(²H,¹³C)methyl-3-oxo-4-(²H₃)butanoicacid (formula 10), 2-hydroxy-2-(²H₃)methyl-3-oxo-4-(²H,¹³C)butanoic acid(formula 11), and 2-(²H₅)ethyl-2-hydroxy-3-oxo-4-(²H,¹³C)butanoic acid(formula 12).
 12. A process for manufacturing a compound of formula I:

the process comprising: a) alkylation, with a methyl group whose carbonatom is ¹³C or ¹²C and whose hydrogen atoms are each independently ¹H or²H (D), or an ethyl group whose carbon atoms are each independently ¹³Cor ¹²C and whose hydrogen atoms are each independently ¹H or ²H (D), ofa 3-oxobutanoate compound whose hydroxyl group in position 1 isprotected by a protecting group, to obtain an intermediate compound, b)hydroxylation of the intermediate compound, c) optionally deprotectionand exchange of one or more ¹H atoms by ²H atoms, wherein thehydroxylation is performed using dimethyldioxirane in the presence ofNickel (II) ions.
 13. A process for analyzing a protein by NMRcomprising the process of claim
 1. 14. A process for analyzing a proteinby NMR comprising labeling the protein with a compound having a formulaI:

wherein: X is ¹H or ²H (D), each Y is independently ¹²C or ¹³C, R¹ is amethyl group whose carbon atom is ¹³C or ¹²C and whose hydrogen atomsare each independently ¹H or ²H (D), R² is (i) a methyl group whosecarbon atom is ¹³C or ¹²C and whose hydrogen atoms are eachindependently ¹H or ²H (D), or (ii) an ethyl group whose carbon atomsare each independently ¹³C or ¹²C and whose hydrogen atoms are eachindependently ¹H or ²H (D), with the provisos that: 1) the hydrogenatoms of the compound are not all, at the same time, either ¹H or ²H(D), and 2) the hydrogen atoms of R¹ and the hydrogen atoms of R² arenot all, at the same time, either ¹H or ²H (D).
 15. The process of claim14, with the additional proviso that: when all the hydrogen atoms of R¹are ²H, then the carbon atoms, in formula I, are not all, at the sametime, ¹²C.
 16. The process of claim 1, which labels Valine.
 17. Theprocess of claim 1, which labels Leucine.
 18. The process of claim 1,which labels Isoleucine.
 19. The process of claim 1, which labels methylgroups of Leucine and Valine.
 20. The process of claim 1, which labelsγ2-methyl groups of Isoleucine.